Phenotypic variation for SY of an association panel of B. napus
SY ranged from 1.51 to 25.07 g/ plant (16.60-fold) in Trial 1 and from 2.18 to 35.88 g/ plant (16.45-fold) in Trial 2 (Table 1). A large range of variations were detected, with the coefficient of variation (CV) varying from 33.71% in Trial 2 to 46.12% in Trial 1 (Table 1). SY in both trials showed an approximate normal distribution (Supplementary Fig. 1). The correlation coefficient (r) of SY between Trial 1 and Trial 2 were 0.41 (Table 1, Supplementary Fig. 2).
Table 1
Mean, maximum (max), minimum (min), range and coefficient of variation (CV, %) of the seed yield (SY) in an association panel of B. napus in Trial 1 and Trial 2
| Seed yield (g/plant) | | CV (%) |
Trials | Mean | Max | Min | Range | |
Trial 1_1 | 5.68 | 13.38 | 1.51 | 11.87 | | 48.75% |
Trial 1_2 | 9.92 | 21.71 | 1.85 | 19.86 | | 44.19% |
Trial 1_3 | 12.31 | 25.07 | 3.1 | 21.97 | | 38.63% |
Trial 1_mean value | 9.34 | 23.35 | 1.53 | 21.83 | | 32.49% |
Trial 2_1 | 17.06 | 34.47 | 2.18 | 32.39 | | 42.85% |
Trial 2_2 | 16.31 | 31.15 | 2.85 | 28.30 | | 36.65% |
Trial 2_3 | 16.96 | 35.88 | 2.18 | 33.7 | | 44.09% |
Trial 2_mean value | 16.79 | 35.79 | 2.4 | 33.38 | | 32.86% |
GWAS of SY of B. napus
GLM and MLM approaches were used to perform GWAS analysis to reveal SNPs significantly associated with SY of B. napus (Supplementary Fig. 3–4, Supplementary Table 2–3). A total of 1773 SNPs were identified to be significantly associated with SY across two trials by GLM approach (P < 6.25 × 10− 07) (Supplementary Table 2). The PVE of 1773 SNPs ranged from 5.57–12.73% (Supplementary Table 2). Thirteen, 20, 19, 34, 38, 92, 390 and 1167 significant SNPs were associated with SY in Trial 1_1, Trial 1_2, Trial 1_3, Trial 1_mean value, Trial 2_1, Trial 2_2, Trial 2_3 and Trial 2_mean value, respectively, and distributed on 19 B. napus chromosomes (Supplementary Fig. 3, Supplementary Table 2). Chromosomes C09 and A09 had the largest (404) and the second largest (281) number of significant SNPs, respectively (Supplementary Table 2). Both MLM and GLM analysis identified the same 16 significant SNPs located on chromosomes A02, A05, A09, A10, C03, C04, C06, explaining 9.78–12.08% of PVE (Supplementary Table 3).
Candidate gene association analysis and haplotype analysis of BnaA01g17200D and BnaA02g08680D
The SNP of chrA01__8920351 co-located on chromosome A01 were significantly associated with SY in the both Trial 2_2 and Trial 2_mean value (Fig. 1a, Supplementary Table 2, 4). Within the 300 kb up/downstream of chrA01__8920351, a total of 87 genes were found (Supplementary Table 5). Previously, transcriptome sequencing was performed on extremely high and low-yield B. napus cultivars to mine yield related candidate genes (Lu et al., 2017). We compared the GWAS results in this study with previous transcriptome data and results show that BnaA01g17200D was the only one significantly differentially expressed gene among the 87 candidate genes on A01 chromosome (Table 2, Supplementary Table 5). The expression level of BnaA01g17200D in bud on the primary branch of high-yield cultivars was significantly higher than that of low-yield cultivars (Table 2, Supplementary Table 5). We extracted the SNPs within the coding region and promoters (upstream, 2000 bp) of BnaA01g17200D, and eight SNPs were located within the 2 kb promoter region and the entire coding region of BnaA01g17200D (Supplementary Table 6). Four SNPs in BnaA01g17200D were detected to be significantly associated with SY (P < 0.001) (Table 3). Further analyzed the four significant SNPs showed that, the T allele of chrA01__9042710, T allele of chrA01__9042762, A allele of chrA01__9044014 and T allele of chrA01__9044072 were high-seed yield alleles (Fig. 1b-f). According to the above four SNPs, two major haplotypes were detected, and cultivars with BnaA01g17200Hap1 (TTAT) had much higher SY than cultivars with BnaA01g17200Hap2 (CAGG) (Fig. 1g).
Table 2
Common candidate genes identified by GWAS and transcriptome sequencing
Gene ID | Log2 (fold change) | P value | Arabidopsis homologue | Annotations | Expression sites |
BnaA01g17200D | 1.97 | 5.62E-16 | AT4G27140 | seed storage albumin 1 | Bud on the primary branch |
BnaA02g08680D | -6.14 | 1.05E-08 | AT2G30480 | hypothetical protein | Bud on the main inflorescence |
Table 3
List of synonymous and non-synonymous SNP variants identified in the candidate genes of BnaA01g17200D, BnaA02g08680D and BnaA09g10430D.
Candidate genes | SNP | Major allele | Minor allele | SNP types | Amino acid changes |
BnaA01g17200D | chrA01__9042710 | T | C | Promoter | \ |
| chrA01__9042762 | T | A | Promoter | \ |
| chrA01__9044014 | A | G | Promoter | \ |
| chrA01__9044072 | T | C | Promoter | \ |
BnaA02g08680D | chrA02__4290037 | G | A | Promoter | \ |
| chrA02__4290087 | G | A | Promoter | \ |
| chrA02__4290091 | C | T | Promoter | \ |
| chrA02__4290092 | T | A | Promoter | \ |
BnaA09g10430D | chrA09__5322341 | C | T | Exon | Arginine to Cysteine |
| chrA09__5322518 | C | T | Intron | \ |
| chrA09__5322553 | T | C | Intron | \ |
| chrA09__5322559 | C | G | Intron | \ |
| chrA09__5322560 | T | G | Intron | \ |
| chrA09__5322565 | A | G | Intron | \ |
| chrA09__5322568 | G | A | Intron | \ |
| chrA09__5322918 | C | T | Intron | \ |
| chrA09__5322966 | A | G | Intron | \ |
| chrA09__5323050 | A | G | Exon | Isoleucine to Valine |
| chrA09__5323064 | T | A | Exon | Leucine to Terminator |
| chrA09__5323081 | A | C | Exon | Glutamine to Histidine |
| chrA09__5323251 | T | A | Promoter | \ |
| chrA09__5323456 | C | T | Promoter | \ |
| chrA09__5323496 | T | C | Promoter | \ |
| chrA09__5323633 | G | C | Promoter | \ |
| chrA09__5323759 | A | C | Promoter | \ |
| chrA09__5324271 | T | C | Promoter | \ |
| chrA09__5324376 | A | G | Promoter | \ |
The SNP of chrA02__4555979 on chromosome A02 was identified in Trial 1_2 and Trial 1_mean value (Fig. 2a, Supplementary Table 2, 4). Based on the LD decay (300 kb), 108 genes were selected within the 300 kb up/downstream of the chrA02__4555979 (Supplementary Table 6). The expression of BnaA02g08680D was 6.14-fold between high-yield B. napus cultivars and low-yield B. napus cultivars in bud on the main inflorescence (Lu et al., 2017, Table 2). Ten SNPs were extracted within the coding region and promoters (upstream, 2000 bp) of BnaA02g08680D and four SNPs in BnaA02g08680D were significantly associated with the SY, and the G allele of chrA02__4290037, G allele of chrA02__4290087, C allele of chrA02__4290091 and T allele of chrA02__4290092 were the high-seed yield alleles (Fig. 2b-f, Supplementary Fig. 6). The above significant SNPs revealed two major haplotypes, and cultivars harboring BnaA02g08680Hap1 (GGCT) had significantly greater seed yield than cultivars harboring BnaA02g08680Hap2 (AATA) (Fig. 2g).
Comparison Of The Significant Snps Associated With Sy With Qtls For Sy
Based on the Darmor-bzh reference genome, a total of 783 SNPs were co-located with the intervals of 47 QTLs for SY in the previous linkage analysis of the B. napus segregation populations (Bouchet et al., 2014; Cai et al., 2016; Luo et al., 2017; Supplementary Table 8). A significant SNP (chrA09__5160639) identified in Trial 2_3 was co-located with the interval of ‘qSY38’ QTL and a gene (BnaA09g10430D) reported to control SY was within the interval of this QTL (Luo et al., 2017, Supplementary Table 8). OsNPF7.2, the orthologue of BnaA09g10430D, regulates tiller number and grain yield in rice (Wang et al., 2018). Candidate gene association analysis of BnaA09g10430D with SNP from the 2-kb promoter and the entire coding region showed that 19 SNPs were significantly associated with SY, and had strong LD with each other (Fig. 3b, Table 3, Supplementary Table 6). Among them, 7 were located in the promoter, 4 in the exon and 8 in the intron region (Table 3). Notably, chrA09__5322341 (C/T), chrA09__5323050 (A/G), chrA09__5323064 (T/A) and chrA09__5323081 (A/C) were located in the exon region of the gene BnaA09g10430D and resulted in amino acid changes from arginine to cysteine, isoleucine to valine, leucine to terminator and glutamine to histidine, respectively (Table 3). Further analysis demonstrated that the T allele of chrA09__5322341 and G allele of chrA09__5323050, A allele of chrA09__5323064, C allele of chrA09__5323081, A allele of chrA09__5323251, T allele of chrA09__5323456, C allele of chrA09__5323496, C allele of chrA09__5323633, C allele of chrA09_5323759, T allele of chrA09__5324271, and G allele of chrA09_5324376 were associated with high SY (Fig. 3c-m). Two major haplotype groups were obtained according to the above 11 significant SNPs (Fig. 3n). Among them, BnaA09g10430Hap2 (TGACATCCCCG) had significantly higher SY than BnaA09g10430Hap1 (CATATCTGATA), therefore BnaA09g10430Hap2 was considered as the favorable haplotype (Fig. 3n). In addition, a total of 67 B. napus cultivars contained these three favorable haplotypes had higher SY than other B. napus cultivars (Fig. 4).